U.S. patent application number 09/961414 was filed with the patent office on 2002-05-16 for enterotoxin adsorbent, method of adsorptive removal, and adsorption apparatus.
Invention is credited to Fujimoto, Tamiji, Furuyoshi, Shigeo, Hirai, Fumiyasu.
Application Number | 20020058032 09/961414 |
Document ID | / |
Family ID | 18774855 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058032 |
Kind Code |
A1 |
Hirai, Fumiyasu ; et
al. |
May 16, 2002 |
Enterotoxin adsorbent, method of adsorptive removal, and adsorption
apparatus
Abstract
The present invention is directed to an enterotoxin adsorbent
comprising a compound having a log P (P denotes a partition
coefficient in an octanol-water system) value of not less than 2.50
as immobilized on a water-insoluble carrier.
Inventors: |
Hirai, Fumiyasu; (Osaka,
JP) ; Fujimoto, Tamiji; (Osaka, JP) ;
Furuyoshi, Shigeo; (Kobe-shi, JP) |
Correspondence
Address: |
KENYON & KENYON
Suite 700
1500 K Street, N.W.
Washington
DC
20005
US
|
Family ID: |
18774855 |
Appl. No.: |
09/961414 |
Filed: |
September 25, 2001 |
Current U.S.
Class: |
424/140.1 ;
604/5.04 |
Current CPC
Class: |
B01J 20/3092 20130101;
B01J 2220/58 20130101; B01J 20/28078 20130101; B01J 20/3242
20130101; A61M 2202/0445 20130101; B01J 20/3212 20130101; C07K
14/31 20130101; A61M 2202/0445 20130101; B01J 20/3217 20130101;
A61M 2202/0014 20130101; A61M 1/3679 20130101; B01J 20/3248
20130101; B01J 20/3255 20130101; B01D 15/00 20130101; B01J 20/3219
20130101 |
Class at
Publication: |
424/140.1 ;
604/5.04 |
International
Class: |
A61K 039/395; A61M
037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 2000 |
JP |
2000-291830 |
Claims
1. An enterotoxin adsorbent comprising a compound with a log P, in
which P represents a partition coefficient in an octanol-water
system, value of not less than 2.50 as immobilized on a
water-insoluble carrier.
2. The adsorbent according to claim 1 wherein said water-insoluble
carrier is a water-insoluble porous carrier.
3. The adsorbent according to claim 2 wherein said water-insoluble
porous carrier has a molecular weight of exclusion limit of 5000 to
600000 for globular protein.
4. A method for adsorptive removal of an enterotoxin in a body
fluid which comprises contacting an enterotoxin-containing body
fluid with the adsorbent according to claim 1.
5. An enterotoxin adsorption apparatus wherein a housing has an
inlet and an outlet for a body fluid as well as a means for
precluding flowing out of an adsorbent therefrom, and is packed
therein the adsorbent according to claim 1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an enterotoxin adsorbent, a
method for adsorptive removal of an enterotoxin, and an adsorption
apparatus comprising said adsorbent as packed in a housing.
BACKGROUND OF THE INVENTION
[0002] Enterotoxins are toxins produced by Staphylococcus aureus,
among other bacteria, which have various biological activities such
as emetic, pyrogenic and mitogenic activities, inducing symptoms of
food poisoning or being causative of toxic shock syndrome
(TSS).
[0003] Staphylococci are broadly distributed in the skin, nasal
cavity, oral cavity, throat, urinary organs and intestinal canal of
various animals inclusive of man as well as in the air, sewage
water, river, foods and so forth and encompass a broad spectrum of
species. Among such numerous species of staphylococci, the one
pathogenic to human beings is Staphylococcus aureus (hereinafter
referred to briefly as S. aureus) which is a coagulase-positive
bacterium. S. aureus induces various infectious diseases and can be
a causative factor in nosocomial infections, thus being of social
concern.
[0004] As the enterotoxin produced by S. aureus, the following 10
species are known to this day: staphylococcal enterotoxins A, B,
C1, C2, C3, D, E, G, HandI (hereinafter referredtobriefly as SEA,
SEB, SEC1, SEC2, SEC3, SED, SEE, SEG, SEH and SEI,
respectively).
[0005] Enterotoxins are known to have superantigen activity. The
ordinary antigen is taken up by the antigen-presenting cell and the
antigen fragments available on fragmentation (conversion to
peptides of 10 to 15 amino acids) are presented, in the form bound
to the pockets of MHC (major histocompatibility complex) class II
molecule, on the surface of the antigen-presenting cell. These
fragments are recognized by the TCR (T cell receptor) .alpha.- and
.beta.-chains of certain T cell clones, whereby the T cells are
activated to set an immune reaction going. On the other hand, in
the case of a superantigen, the antigen is not fragmented but
directlybound to the MHC class II molecule on an antigen-presenting
cell, and then the complex is recognized by TCR on the T cell to
thereby activate the T cell. In this process, the antigen is
recognized by the V.beta. region of the TCR but unlike in the case
of an ordinary antigen, the superantigen is recognized by
substantially the entire population of T cells expressing the
specific V.beta. region to induce activation of the T cells and,
hence, production of cytokines. Thus, in an individual exposed to a
superantigen, an enormous population of T cells is activated as
compared with the ordinary specific immune response to consequently
release cytokines within a brief time, thus being suspected to
induce abnormal reactions of the living body.
[0006] By using a specific antibody against an enterotoxin, an MHC
class II protein or the like, the enterotoxin can be removed from a
body fluid such as blood, plasma or serum, a culture supernatant, a
foodstuff or a beverage but such antibodies are not only expensive
but have the drawback that sterilization causes denaturation and
serious decreases in adsorptive capacity.
[0007] Therefore, the advent has been awaited of an enterotoxin
adsorbent which may be produced easily at low cost and will be
highly effective.
[0008] Incidentally, Japanese Kokai Publication Hei-10-290833
discloses an adsorbent for TSST-1 (toxic shock syndrome toxin-1)
comprising a compound having a log P (P denotes a partition
coefficient in an octanol-water system) value of not less than 2.50
as immobilized but the literature is reticent about adsorption of
an enterotoxin.
SUMMARY OF THE INVENTION
[0009] The object of the present invention is to provide an
adsorbent with which enterotoxins in body fluids can be efficiently
adsorbed and removed, a method for adsorptive removal of an
enterotoxin from a body fluid which comprises using said adsorbent,
and an enterotoxin adsorption apparatus.
[0010] The inventors of the present invention explored in earnest
for an adsorbent which may be capable of removing enterotoxins from
body fluids with good efficiency. As a result, they discovered that
the enterotoxin occurring in a body fluid can be efficiently
adsorbed and removed with an adsorbent comprising a compound having
a log P value of not less than 2.50 as immobilized on a
water-insoluble carrier. The present invention has been developed
on the basis of the above finding.
[0011] The present invention, therefore, is directed to an
enterotoxin adsorbent comprising a compound with a log P, in which
P represents a partition coefficient in an octanol-water system,
value of not less than 2.50 as immobilized on a water-insoluble
carrier.
[0012] The present invention is further directed to a method for
adsorptive removal of an enterotoxin in a body fluid
[0013] which comprises contacting an enterotoxin-containing body
fluid with an enterotoxin adsorbent,
[0014] said adsorbent comprising a compound with a log P, in which
P represents a partition coefficient in an octanol-water system,
value of not less than 2.50 as immobilized on a water-insoluble
carrier.
[0015] The present invention is further directed to an enterotoxin
adsorption apparatus
[0016] wherein a housing has an inlet and an outlet for a body
fluid as well as a means for precluding flowing out of an adsorbent
therefrom, and is packed therein an enterotoxin adsorbent,
[0017] said adsorbent comprising a compound with a log P, in which
P represents a partition coefficient in an octanol-water system,
value of not less than 2.50 as immobilized on a water-insoluble
carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic cross-section view showing an
enterotoxin adsorption apparatus embodying the principles of the
invention.
[0019] FIG. 2 is a diagrammatic representation of the relation
between flow rate and pressure loss as determined for 3 kinds of
carrier materials.
EXPLANATION OF NUMERIC SYMBOLS
[0020] 1. body fluid inlet
[0021] 2. body fluid outlet
[0022] 3. enterotoxin adsorbent
[0023] 4. and 5. filter which allows passage of a body fluid but
does not allow passage of said enterotoxin adsorbent
[0024] 6. column
[0025] 7. enterotoxin adsorption apparatus
DETAILED DESCRIPTION OF THE INVENTION
[0026] The enterotoxin in the context of the present invention is a
toxin comprising a soluble protein having a molecular weight of
25,000 to 30,000 as produced by S. aureus.
[0027] The body fluid includes blood, plasma, serum, ascites,
lympha, synovial fluid, fractions or components of any of them, and
other humoral biological materials.
[0028] The log P value is a parameter of hydrophobicity of a
compound and the partition coefficient P in the representative
octanol-water system is determined in the following manner. Thus,
the compound of interest is first dissolved in octanol (or water),
an equal quantity of water (or octanol) is then added, and the
mixture is shaken with Griffin flask shaker (manufactured by
Griffin & George, Ltd.) for 30 minutes. The mixture is then
centrifuged at 2000 rpm for 1 to 2 hours and the concentrations of
the compound in the octanol layer and the water layer are measured
spectrometrically or by GLC at room temperature and atmospheric
pressure, among other techniques. Then, the P value is calculated
by means of the following equation.
[0029] P=Coct/Cw Coct: concentration of the compound in octanol
layer Cw: concentration of the compound in water layer
[0030] The adsorbent of the invention comprises a water-insoluble
carrier and, as supported thereon, a compound having a log P value
of not less than 2.50 as determined by the above method.
[0031] The log P values of various compounds have so far been
measured by many workers and such measured log P values have been
compiled by C. Hansch et al. [Partition Coefficients and Their
Uses; Chemical Reviews 71, 525 (1971)].
[0032] Referring to compounds with unknown measured log P values,
the values (.SIGMA.f) calculated using the hydrophobic fragmental
constant f shown in The Hydrophobic Fragmental Constant, Elsevier
Sci. Pub. Com., Amsterdam (1977) can be used as a reference. The
hydrophobic fragmental constant is a value representing the
hydrophobicity of various fragments as determined statistically
based on a large number of measured log P values. The sum of f
values of various fragments constituting a compound is
approximately equal to the log P value. The term log P as used in
this invention for any compound with an unknown log P value means
the .SIGMA.f value of the compound.
[0033] In the screening for compounds effective for adsorption of
enterotoxins, compounds having various log P values were
respectively immobilized on a water-insoluble carrier and evaluated
for their adsorptive affinity for enterotoxins. As a result, it was
found that compounds having log P values not smaller than 2.50,
preferably not smaller than 2.80, more preferably not smaller than
3.00, are effective in the adsorption of enterotoxins, while
compounds with log P values smaller than 2.50 do not appreciably
adsorb enterotoxins. By way of illustration, assuming that an
alkylamine is immobilized as said compound on a water-insoluble
carrier, the change from n-hexylamine (log P=2.06) to n-octylamine
(log P=2.90) leads to a phenomenal increase in the adsorptive
affinity for enterotoxins. These findings suggest that the
adsorption of an enterotoxin by the adsorbent of the invention is
attributable to the hydrophobic interaction between the enterotoxin
and the atomic group introduced onto the carrier by immobilization
of a compound having a log P value of not less than 2.50 and that
any compound having a log P value of less than 2.50 is too low in
hydrophobicity to exhibit a sufficiently high adsorptive affinity
for enterotoxins.
[0034] The compound that can be immobilized on a water-insoluble
carrier with advantage in the practice of the invention is not
particularly restricted only provided that its log P value is not
smaller than 2.50. However, when a compound is immobilized on a
carrier by a chemical coupling reaction, a portion of the compound
is eliminated in many instances and in case the eliminated group
contributes a great deal to the overall hydrophobicity of the
compound, that is to say the hydrophobicity of the atomic group
immobilized on the carrier is reduced to a .SIGMA.f value of less
than 2.50 due to the eliminated group, the particular compound is
not suitable in light of the object and spirit of the invention. A
case in point is the immobilization of isopentyl benzoate
(.SIGMA.f=4.15) on a hydroxyl group-containing carrier by a
transesterification reaction. In this case, the atomic group
actually immobilized on the carrier is C.sub.6H.sub.5CO-- and this
atomic group has a .SIGMA.f value of less than 1. Whether a
compound of this type is suitable as the compound for use in the
invention can be simply found by checking to see whether the
compound obtainable by substituting hydrogen for the eliminated
group has a log P value of not less than 2.50.
[0035] The preferred, among compounds having log P values not
smaller than 2.50, are unsaturated hydrocarbons, alcohols, amines,
thiols, carboxylic acids and derivatives thereof, halides,
aldehydes, hydrazides, isocyanates, oxirane ring-containing
compounds such as glycidyl ethers, halogenated silanes, and other
compounds having functional groups useful for bonding to the
carrier. As representative examples of such compounds, there can be
mentioned amines such as n-heptylamine, n-octylamine, decylamine,
dodecylamine, hexadecylamine, octadecylamine, 2-aminooctene,
naphthylamine, phenyl-n-propylamine, diphenylmethylamine, etc.;
alcohols such as n-heptyl alcohol, n-octyl alcohol, dodecyl
alcohol, hexadecyl alcohol, 1-octen-3-ol, naphthol,
diphenylmethanol, 4-phenyl-2-butanol, etc. and glycidyl ethers of
such alcohols; carboxylic acids such as n-octanoic acid, nonanoic
acid, 2-nonenoic acid, decanoic acid, dodecanoic acid, stearic
acid, arachidonic acid, oleic acid, diphenylacetic acid,
phenylpropionic acid, etc. and the corresponding acid halides and
derivatives such as esters and amides; halides such as octyl
chloride, octyl bromide, decyl chloride, dodecyl chloride, etc.;
thiols such as octanethiol, dodecanethiol, etc.; halosilanes such
as n-octyltrichlorosilane, octadecyltrichlorosilane, etc., and
aldehydes such as n-octyl aldehyde, n-capryl aldehyde, dodecyl
aldehyde, and so forth.
[0036] Aside from the foregoing compounds, compounds such that the
hydrogen atom in the hydrocarbon moiety of any of the compounds
mentioned by way of example above have been substituted by a
halogen atom, a substituent group containing a hetero atom such as
N, O or S, a different alkyl group or the like and having log P
values not smaller than 2.50, as well as compounds having log P
values not less than 2.50 among the compounds listed in the above
general review of C. Hansch et al. [Partition Coefficients and
Their Uses: Chemical Reviews 71, 525 (1971), pages 555-613] can
also be mentioned. For use in the invention, these compounds are
not exclusive choices.
[0037] These compounds may be used each independently or in a
combination of two or more species, optionally even in combination
with a compound having a log P value smaller than 2.50.
[0038] The water-insoluble carrier as a constituent of the
adsorbent of the invention is a material which is solid at
atmospheric temperature and pressure and only sparingly soluble in
water.
[0039] The shape of the water-soluble carrier in the present
invention may for example be granular, sheet-like, filamentous or
hollow fiber-like, although these are not exclusive choices. The
size of the carrier is not particularly restricted, either.
[0040] The water-insoluble carrier as a constituent of the
adsorbent of the invention includes inorganic matrices such as
glass beads, silica gel, etc.; organic matrices such as synthetic
polymers, e.g. crosslinked polyvinyl alcohol, crosslinked
polyacrylates, crosslinked polyacrylamide, crosslinked polystyrene,
etc. and polysaccharides, e.g. crystalline cellulose, crosslinked
cellulose, crosslinked agarose, crosslinked dextrin, etc.; and
composite matrices comprising organic-organic, organic-inorganic,
or other combinations of the above-mentioned matrices, to mention
typical examples.
[0041] Among these, hydrophilic matrices are preferred because of
their comparatively low nonspecific adsorption and good adsorption
selectivity for enterotoxins. The term "hydrophilic carrier" is
used herein to mean a carrier such that when the compound
constituting the carrier is made into a flat plate, the angle of
contact between it and water is not greater than 60 degrees.
Various techniques are known for measurement of the contact angle
of water but as described by Ikeda in his book Jikken Kagaku Sensho
(Selected readings in Experimental Chemistry), Chemistry of
Colloids, Chapter 4, Thermodynamics of Interfaces, pp. 75 to 104,
Mokabo (1986), the most common method comprises placing a drop of
water on a flat plate made of the compound and measuring the angle
of contact with water. The compound giving such an angle of contact
not greater than 60 degrees as measured by the above method
includes cellulose, polyvinyl alcohol, saponified ethylene-vinyl
acetate copolymer, polyacrylamide, polyacrylic acid,
polymethacrylic acid, poly(methyl methacrylate), polyacrylic
acid-grafted polyethylene, polyacrylamide-grafted polyethylene,
glass and so forth.
[0042] These water-insoluble carrier materials preferably have
amultiplicity of pores of suitable size, that is to say aporous
structure. The carrier having a porous structure includes not only
a carrier having spaces (macropores) defined by clusters of
microspheres when a basal carrier polymer forms single spherical
particles by cohesion of microspheres but also a carrier having
micropores formed among the clusters of cores within each
microsphere constituting a basal polymer carrier and a carrier
having micropores formed when a copolymer having a
three-dimensional structure (a polymer network) is swollen in the
presence of an organic solvent having an affinity for the
polymer.
[0043] Furthermore, in consideration of the adsorptive capacity per
unit volume of the adsorbent, said water-insoluble carrier having a
porous structure is more preferably of the total porosity type than
of the surface porosity type and the void volume and specific
surface area are preferably as large as possible within limits not
detracting from adsorption efficiency.
[0044] As a carrier satisfying these preferred requirements, a
porous cellulose carrier can be mentioned. The porous cellulose
carrier has several meritorious characteristics. Thus, (1) because
it has comparatively high mechanical strength and toughness, this
carrier does not collapse or give dust in stirring and other
operations and even when a body fluid is passed through a column
packed with the carrier at a high speed, the carrier is not
compacted, thus permitting a high flow rate. Furthermore, the
porous structure of the carrier is not easily affected by
high-pressure steam sterilization. (2) Because this carrier is made
up of cellulose, it is hydrophilic, has a large number of hydroxyl
groups available for binding a ligand, and features little
nonspecific adsorption. (3) Even if the void volume is increased,
an adsorptive capacity as large as that of a soft carrier may be
insured because of its comparatively high strength. (4) The carrier
ranks high in safety as compared with synthetic polymer and other
matrices. Therefore, this carrier is one of the most favorable
matrices for use in the present invention, although this is not an
exclusive choice. Moreover, the above-mentioned matrices may be
used each independently or in the form of a mixture of two or more
species.
[0045] More preferably, said water-insoluble carrier having a
porous structure is characterized in that while the adsorption load
may enter its pores with a fairly high probability, the entry of
other proteins is precluded as much as possible. Thus, the
enterotoxin to be adsorbed by the adsorbent of the invention is a
protein having a molecular weight within the range of 25,000 to
30,000 and for efficient adsorption of this protein, the carrier is
preferably such that the enterotoxin may find its way into its
porous structure in a large ratio but other proteins are prevented
from entering the pores. As a molecular weight marker of a
substance capable of entering a porous structure, the molecular
weight of exclusion limit is generally used. As described in
several books (e.g. Hatano Hiroyuki & Hanai Toshihiko:
Experimental high Performance Liquid Chromatography, Kagaku Dojin),
the molecular weight of exclusion limit means the molecular weight
of the smallest of the molecules prevented from entering the pores
(entry rejected) in gel permeation chromatography. Molecular
weights of exclusion limit have been well documented generally for
globular proteins, dextran, polyethylene glycol, etc. and in the
case of the carrier for use in the invention, it is appropriate to
use the value found for globular proteins.
[0046] Investigations undertaken by the inventors using matrices
varying in molecular weight of exclusion limit revealed that the
range of molecular weights of exclusion limit for globular proteins
which is suitable for adsorption of enterotoxins is 5,000 to
600,000. Thus, when a carrier having a molecular weight of
exclusion limit of less than 5000 for globular protein is employed,
the amount of adsorption of enterotoxins is too small to endorse
its practical utility. On the other hand, when the molecular
weight- of exclusion limit of 600,000 is exceeded, the adsorption
of proteins (mostly albumin) other than enterotoxins is increased
so that the practical utility of the carrier is low in terms of
selectivity. Therefore, the preferred range of molecular weights of
exclusion limit for globular protein as the carrier in the present
invention is 5,000 to 600,000, with the range of 6,000 to 400,000
being the more preferred and the range of 10,000 to 300,000 being
particularly preferred.
[0047] Furthermore, the carrier preferably has a functional group
which can be used for the ligand-binding reaction. The functional
group mentioned above includes but is not limited to hydroxyl,
amino, aldehyde, carboxyl, thiol, silanol, amido, epoxy, halogen,
succinylimino, and acid anhydride.
[0048] The carrier which can be used in the present invention
includes both a rigid carrier and a soft carrier. However, when it
is used as a constituent of the adsorbent for extracorporeal
circulation, it is important that when the adsorbent is packed into
a column and the body fluid is passed through it, no plugging
should take place. To ensure this, a sufficient mechanical strength
is required of the carrier. Therefore, the carrier for use in the
invention is more preferably a rigid carrier. The rigid carrier
mentioned above means a carrier such that, taking a granular
carrier as an example, when a cylindrical column is evenly packed
with the carrier and an aqueous fluid is passed, the relation
between pressure loss .DELTA.P and flow rate is linear up to 0.3
kg/cm.sup.2 as described hereinafter in Reference Example.
[0049] While the adsorbent of the invention can be obtained by
immobilizing a compound having a log P value of not less than 2.50
on a water-insoluble porous carrier, various known techniques can
be liberally used for the immobilization. However, when the
adsorbent of the invention is used for extracorporeal circulation
therapy, it is important from safety points of view to minimize the
risk of elimination and elution of the ligand during sterilization
and therapy and, in this sense, immobilization by covalent bonding
is preferred.
[0050] Various techniques are available for the adsorptive removal
of an enterotoxin from body fluids by means of the adsorbent of the
invention. The simplest method comprises withdrawing a body fluid
into a bag or the like, mixing the adsorbent with the body fluid to
adsorptively remove enterotoxins, and filtering off the adsorbent
to recover the fluid having eliminated the enterotoxins. Another
method comprises packing the adsorbent into a housing equipped with
an inlet and an outlet for a body fluid and, disposed at the
outlet, further with a filter which allows passage of a body fluid
but intercepts the adsorbent and passing the body fluid through the
housing. Whichever desired of the methods can be utilized but the
latter method is not only expedient but, when the system is built
into an extracorporeal circuit, enables one to remove enterotoxins
from a patient's body fluid, particularly the blood, on line and
with good efficiency. The adsorbent of the invention is suitable
for this method.
[0051] In the extracorporeal circuit mentioned above, the adsorbent
of the invention may be used independently but may be used in
combination with a different extracorporeal therapeutic system. As
an example of such combination, an artificial dialysis circuit can
be mentioned. Thus, the adsorbent can be used in conjunction with a
dialysis treatment.
[0052] The enterotoxin adsorption apparatus of the invention, which
makes use of the above-described adsorbent of the invention, is now
described with reference to FIG. 1 which is a schematic
cross-section view showing an embodiment. In FIG. 1, the reference
numeral 1 represents a body fluid inlet, 2 a body fluid outlet, 3
an enterotoxin adsorbent according to the invention, 4 and 5 each a
filter which allows passage of the body fluid and its components
but does not allow passage of said enterotoxin adsorbent, 6 a
column, and 7 an enterotoxin adsorption apparatus. It should,
however, be understood that the enterotoxin adsorption apparatus is
not restricted to such a specific embodiment but may be any
apparatus comprising a housing equipped with an inlet and an outlet
for a fluid and a means for precluding flowing out of an
enterotoxin adsorbent therefrom and, as packed therein, said
enterotoxin adsorbent.
[0053] The means for precluding flowing out of the adsorbent
includes a wire-mesh filter, a nonwoven cloth filter, a cotton pad
filter, and so forth. The housing is not particularly restricted in
geometry, material or size but one having a columnar configuration
is preferred. The preferred housing material withstands a
sterilization procedure, thus including silicon-coated glass,
polypropylene, poly(vinyl chloride), polycarbonate, polysulfone,
polymethylpentene, and so forth. The capacity and size of the
housing are preferably 50 to 1500 ml and 2 to 20 cm in diameter,
more preferably 100 to 800 ml and 3 to 15 cm in diameter, still
more preferably 150 to 400 ml and 4 to 10 cm in diameter.
[0054] By means of the adsorbent comprising a compound having a log
P value of not less than 2.50 as immobilized on a water-insoluble
carrier according to the invention, enterotoxins can be adsorbed
and removed from body fluids with good efficiency.
EXAMPLES
[0055] The following examples are intended to describe the present
invention in further detail and should by no means be construed as
defining the scope of the invention.
[0056] [Reference Example]
[0057] A glass-made cylindrical column (9 mm in. dia..times.150 mm
long) fitted with a 15 .mu.m (pore size) filter at both ends was
uniformly filled with agarose material (product of Bio-Rad; Biogel
A-5m, particle diameter 50 to 100 mesh), vinyl polymer material
(product of Tosoh Corporation; Toyopearl HW-65, particle diameter
50 to 100 .mu.m) and cellulose material (product of Chisso
Corporation; Cellulofine GC-700m, particle diameter 45 to 105
.mu.m) . Using a peristaltic pump, water was caused to flow through
the column and the relation between flow rate and pressure loss
.DELTA.P was determined. The result is shown in FIG. 2.
[0058] It is apparent from FIG. 2 that whereas the flow rate
increased in substantial proportion withan increasing pressure in
the cases of Toyopearl HW-65 and Cellulofine GC-700m, the flow rate
failed to increase owing to compaction even when the pressure was
increased in the case of Biogel A-5m. For the purposes of the
present invention, any carrier, such as the former two materials,
which gives a linear relation between pressure loss .DELTA.P and
flow rate up to 0.3 kg/cm.sup.2 is defined as a rigid carrier.
Example 1
[0059] To 170 ml of the cellulosic porous carrier Cellulofine
GC-200m (product of Chisso Corporation; molecular weight of
exclusion limit for globular protein: 140,000) was added sufficient
water to make 340 ml. Then, 90 ml of 2 M sodium hydroxide/H.sub.2O
was added and the temperature was adjusted to 40.degree. C. To this
was added 31 ml of epichlorohydrin, and the reaction was carried
out at 40.degree. C. with stirring for 2 hours. After completion of
the reaction, the reaction product was rinsed well with water to
give epoxidized Cellulofine GC-200m.
[0060] To 10 ml of this epoxidized Cellulofine GC-200m was added
200 mg of n-hexadecylamine (.SIGMA.f=7.22), and the reaction was
conducted in ethanol under stationary conditions at 45.degree. C.
for 6 days. After completion of the reaction, the reaction product
was washed thoroughly with ethanol and water in the order mentioned
to give n-hexadecylamine-immobilized Cellulofine GC-200m.
[0061] To 0.5 ml of the above n-hexadecylamine-immobilized
Cellulofine GC-200m was added 3 ml of fetal bovine serum (FBS)
containing about 600 pg/ml each of 3 different enterotoxins, namely
SEA, SEB, and SEC1, and the mixture was shaken at 37.degree. C. for
2 hours. After 2 hours, the adsorbent was separated from the
supernatant and the concentrations of the respective enterotoxins
in the supernatant were determined by ELISA.
[0062] The ELISA of each enterotoxin was carried out as follows.
The primary antibody rabbit anti-SEA (or SEB or SEC) IgG (product
of Toxin Technology) was diluted with a coating buffer and
distributed onto amicrotiter plate, 100 .mu.l per well. After
overnight standing at 4.degree. C., the microtiter plate was washed
and 3% bovine serum albumin solution was added, 200 .mu.l per well.
The plate was allowed to sit again at room temperature for 2 hours
and, then, washed. Thereafter, the standard solutions of the
respective enterotoxins and the supernatants before and after
incubation were placed in a 100 .mu.l microtiter plate. After 2
hours of sitting at room temperature, the plate was washed. The
secondary antibody rabbit anti-SEA (or SEB or SEC) HRP (product of
Toxin Technology) was diluted with 1% bovine serum albumin solution
and added, 100 .mu.l/well. After 2 hours of sitting at room
temperature, the plate was washed. Then, 100 .mu.l/well of
o-phenylenediamine solution was added and the plate was allowed to
sit at room temperature for 10 minutes. Thereafter, 100 .mu.l/well
of 4 N-sulfuric acid was added and the absorbance at 492 nm was
measured. By comparison with the absorbance of the standard
solution, the concentration of each enterotoxin was estimated.
Example 2
[0063] To 10 ml of the epoxidized Cellulofine GC200m obtained in
Example 1 was added 200 mg of n-octylamine (log P=2.90), and the
reaction was conducted in 50 (v/v) % ethanol/water at 45.degree. C.
under stationary conditions for 6 days. After completion of the
reaction, the reaction product was washed thoroughly with 50 (v/v)
% ethanol/water, ethanol, 50% (v/v) % ethanol/water, and water in
the order mentioned to give n-octylamine-immobilized Cellulofine
GC200m.
[0064] The above n-octylamine-immobilized Cellulofine GC200m was
shaken with FBS containing 3 kinds of enterotoxins just as in
Example 1. The adsorbent was separated from the supernatant and the
concentration of each enterotoxin in the supernatant was determined
by ELISA.
Comparative Example 1
[0065] Using n-hexylamine (log P=2.06) in lieu of n-octylamine, the
procedure of Example 2 was otherwise faithfully repeated to give
n-hexylamine-immobilized Cellulofine GC200m. This
n-hexylamine-immobilize- d Cellulofine GC200m was shaken with FBS
containing 3 kinds of enterotoxins just as in Example 1. The
adsorbent was separated from the supernatant and the concentration
of each enterotoxin in the supernatant was determined by ELISA.
Comparative Example 2
[0066] Using n-butylamine (log P=0.97) in lieu of n-octylamine, the
procedure of Example 2 was otherwise faithfully repeated to give
n-butylamine-immobilized Cellulofine GC200m. This
n-butylamine-immobilize- d Cellulofine GC200m was shaken with FBS
containing 3 kinds of enterotoxins just as in Example 1. The
adsorbent was separated from the supernatant and the concentration
of each enterotoxin in the supernatant was determined by ELISA.
Comparative Example 3
[0067] Cellulofine GC-200m was shaken with FBS containing 3 kinds
of enterotoxins as in Example 1. The adsorbent was separated from
the supernatant and the concentration of each enterotoxin in the
supernatant was determined by ELISA.
1 TABLE 1 Concentration of enterotoxin (pg/ml) SEA SEB SEC1 Example
1 150 130 180 Example 2 200 150 210 Comp. Ex. 1 565 590 500 Comp.
Ex. 2 570 600 520 Comp. Ex. 3 580 610 540
* * * * *